The Food and Drug Administration (FDA) regulates the manufacture of test kits for the detection of HIV-2 infection. As part of the regulation of these tests the FDA requires every lot of kits to pass a lot release test. In this study we have used commercial and in house tests to characterize the antibody specificities of prospective sera for use as lot release panel members. Twenty sera were obtained for analysis. Based upon commercial viral lysate tests these sera were categorized into two groups (predominately HIV-2 and dual HIV-1, HIV-2 reactive). All specimens were characterized as having strong gag and env reactivity on HIV-2 RIPA and western blot (WB) assays. Predominantly HIV-2 reactive specimens were characterized as having strong reactivity to env peptides on HIV-2 WB and weak reactivity to gag and env peptides on HIV-1 WB. Dually reactive specimens, in general, gave only slightly stronger gag and env reactivity on HIV-2 WB than HIV-1 WB. These data suggest that reactivity with gag epitopes are primarily responsible for cross reactivity of HIV-2 infected individuals reacting with HIV-1 antigens. Further studies using synthetic peptides with these sera should help to further discriminate between cross reactivity and the possibility of dual infections by identifying the reactive epitopes with genetically conserved epitopes.